Monocytes are stimulated by specific ligands to participate in processes which contribute to inflammation and host defense. Although the mechanisms of monocyte infiltration into tissues are not fully understood, locally generated cytokines, notably monocyte chemotactic peptide 1 (MCP-1), appear to play a crucial role in monocyte recruitment. Two distinct isoforms of the MCP-1 receptor CCR2 that differ only in their alternatively spliced intracellular carboxyl tail have been identified. A major aim of the proposed work is to determine the relative expression of CCR2A and CCR2B on the plasma membrane of freshly isolated human monocytes, and to study the distinct cellular responses they induce. The difference between both isoforms lies exclusively in the amino acid sequence of the carboxy tail which is involved in signal transduction. This difference could alter the selective coupling to GTP binding proteins, resulting in distinct cellular responses. Antibodies directed against specific receptor domains and transfection systems will be used to investigate the interaction between receptor and GTP binding protein, and the resulting cellular responses. Specific aim 2 is to identify and characterize receptor domains essential for ligand binding and signaling. These results will be the basis for the design of antagonists. In preliminary studies, antibodies against the 1st and 2nd extracellular loops prevented ligand binding and inhibited the functional response of monocytes to MCP-1 indicating that multiple domains are involved in receptor function. First, chimeric receptors will be constructed to determine more globally the ligand binding domain(s), and then the microstructures and individual amino acid residues essential for ligand binding and signaling will be determined. In specific aim 3, we will determine the effects of low density lipoproteins (LDL) on expression of CCR2A and CCR2B. In preliminary studies, LDL dramatically increased the expression of CCR2B in monocytic cells lines, resulting in augmented chemotactic response to MCP-1. Experiments will be designed to determine the mechanisms by which LDL regulates CCR2 expression and to study the pathologic effects of hyperresponsive monocytes. We believe our findings will provide fundamental insights into the mechanisms of monocyte activation and recruitment by CCR2, expand our knowledge of various inflammatory diseases processes, including atherosclerosis, and could be used to develop novel and effective therapeutic strategies.